Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS December 17, 1985 Pages 563-573 POLYAMINES APPEAR TO BE SECOND MESSENGERS IN MEDIATING Ca 2+ FLUXES AND NEUROTRANSMITTER RELEASE IN POTASSIUM-DEPOLARIZED SYNAPTOSOMES Zafar Iqbal and Harold Koenig+ Neurology Service, VA Lakeside Medical Center, 333 East Huron Street,Chicago, IL 60611 and Departments of Neurology and Biochemistry, Northwestern University Medical School, Chicago, IL 60611 Received August 28, 1985 High potassium (50 mM) depolarization induces a rapid (~15 set) in- crease in the levels of the polyamines putrescine, spermidine and sper- mine and their rate-regulating synthetic enzyme ornithine decarboxylase in synaptosomes from rat cerebral cortex, The ornithine decarboxylase inhibitor a-difluoromethylomithine blocked the K+-stimulated increase in enzyme activity and polyamines and also suppressed the increase in 45Ca2+ influx and efflux and the Ca2+ -dependent release of GABA and norepinephrine. Added putrescine attenuated or negated the effects of a-difluoromethylornithine. These results suggest that enhanced poly- amine synthesis is required for potassium depolarized stimulation of synaptic function. 0 1985 Academic Press, Inc. An increase in free cytosolic Ca 2+ is considered to be a key step in the release of neurotransmitters from nerve terminals in response to membrane depolarization or the binding of transmitters to receptors (1,2). When nerve terminals are depolarized, the increase in cytosolic Ca 2+ is thought to result from an increased inflow of extracellular Ca 2+ via slow, voltage-dependent Ca 2+ channels in the presynaptic membrane. Under cer- tain circumstances, neurotransmitter release has been attributed to an increase in cytosolic Ca 2+ mobilized from intracellular sites such as mitochondria and endoplasmic reticulum (3). The molecular mechanisms whereby membrane depolarizing stimuli, neurotransmitters and other sur- face agonists increase Ca 2+ inflow across the plasma membrane or regulate -t To whom correspondence should be addressed. Abbreviations: ODC, ornithine decarboxylase; DFMO, a-difluoromethylomi- thine; PSS, physiological salt solution. 0006-291X/85 $1.50 563 Copyright 0 1985 by Academic Press, Inc. AN rights of reproduction in any form reserved.
Transcript
Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
December 17, 1985 Pages 563-573
POLYAMINES APPEAR TO BE SECOND MESSENGERS IN MEDIATING Ca 2+
FLUXES AND NEUROTRANSMITTER RELEASE IN POTASSIUM-DEPOLARIZED SYNAPTOSOMES
Zafar Iqbal and Harold Koenig+
Neurology Service, VA Lakeside Medical Center, 333 East Huron Street,Chicago, IL 60611
and Departments of Neurology and Biochemistry,
Northwestern University Medical School, Chicago, IL 60611
Received August 28, 1985
High potassium (50 mM) depolarization induces a rapid (~15 set) in- crease in the levels of the polyamines putrescine, spermidine and sper- mine and their rate-regulating synthetic enzyme ornithine decarboxylase in synaptosomes from rat cerebral cortex, The ornithine decarboxylase inhibitor a-difluoromethylomithine blocked the K+-stimulated increase in enzyme activity and polyamines and also suppressed the increase in 45Ca2+ influx and efflux and the Ca2+ -dependent release of GABA and norepinephrine. Added putrescine attenuated or negated the effects of a-difluoromethylornithine. These results suggest that enhanced poly- amine synthesis is required for potassium depolarized stimulation of synaptic function. 0 1985 Academic Press, Inc.
An increase in free cytosolic Ca 2+ is considered to be a key step
in the release of neurotransmitters from nerve terminals in response to
membrane depolarization or the binding of transmitters to receptors (1,2).
When nerve terminals are depolarized, the increase in cytosolic Ca 2+ is
thought to result from an increased inflow of extracellular Ca 2+ via slow,
voltage-dependent Ca 2+ channels in the presynaptic membrane. Under cer-
tain circumstances, neurotransmitter release has been attributed to an
increase in cytosolic Ca 2+ mobilized from intracellular sites such as
mitochondria and endoplasmic reticulum (3). The molecular mechanisms
whereby membrane depolarizing stimuli, neurotransmitters and other sur-
face agonists increase Ca 2+ inflow across the plasma membrane or regulate
Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the outflow of Ca2+ from mitochondria and other organelles in nerve
endings are unknown.
Recent studies in this laboratory have shown that a transient in-
crease in activity of omithine decarboxylase (ODC, EC 4.1.1.17), the
first and rate-limiting enzyme of polyamine synthesis, and a subsequent
rise in polyamine levels are early cellular events following receptor
activation by testosterone (4), the f3-adrenergic agonist 1-isoproterenol
(5), the thyroid hormone triiodothyronine (6), and insulin (7). This
polyamine synthesis has been found to be essential for hormonal stimula-
tion of Ca2+ fluxes and the mediation of several Ca 2+
-dependent membrane
transport processes (endocytosis, hexose transport, amino acid transport).
These observations form the basis of a new model for signal transduction
and stimulus-response coupling in which the polyamines putrescine, sper-
midine and spermine serve as intracellular messengers or signals to in-
crease free cytosolic 2+ 2+ 2+ Ca by stimulating Ca influx and mobilizing Ca
from intracellular sites via a cation exchange reaction (4-7). We report
here that depolarization by high K+ provokes a rapid, transient in-
crease in ODC activity and a more sustained increase in polyamine con-
centrations in synaptosomes isolated from rat cerebral cortex. This en-
hanced polyamine synthesis appears to be essential for the depolarization-
mediated changed in Ca 2+ influx and efflux and the Ca 2+-dependent release
of y-aminobutyrate and norepinephrine. Some of these results have appear-
ed in abstracts (8,9).
Materials and Methods
Materials. 45Ca2+, 1-[3-3H(n)-ornithine (20 Ci/mmol), y-[2,3-3H(n)]- aminobutyric acid (38.1 Ci/mmol), 1-[2,5,6-3H]norepinephrine (53.5 Ci/mmol) and Aquasol were obtained from New England Nuclear (Boston, MA). a-Di- fluoromethylornithine was a gift of Merrell-National Laboratories (Cincinnati, OH). Sprague-Dawley rats were from Holtzman (Madison, WI).
Preparation of Synaptosomes. Synaptosomes were isolated from rat cerebral cortex by Ficoll-sucrose density gradient centrifugation (10). The band sedimenting between 8% and 12% Ficoll was removed and diluted with 5 vol. of physiological salt solution (PSS) containing 119 mM NaCl, 4 mM KCl, 1.2 mM MgC12, 0.2 mM CaC12, 10 mM glucose and 20 mM HEPES adjusted to pH 7.4 with Tris base. Synaptosomes were sedimented by centrifugation and resuspended in cold PSS to give a protein concen- tration of 2-3 mg/ml. Electron microscopic examination confirmed that
564
Vol. 133, No. 2, 1985 8lOCHEMlCAL AND
pinched-off nerve endings were the major preparations.
EIOPHYSICAL RESEARCH COMMUNICATIONS
component of these synaptosomal
Incubations of Synaptosomes for Polyamine Synthesis. Synaptosomal samples were preincubated in PSS with appropriate additions at 37O C under 95% 0215% CO2 for 15 min. Incubations were started by adding an equal volume of prewarmed PSS or double-strength depolarizing medium (to give a final concentration of 50 mM K+ and 73 mM Na+) and the same additions. Incubations were terminated by adding 10 vol. of cold PSS. Synaptosomes were sedimented by centrifugation at 5000 rpm for 5 min and suspended either in 0.2 M perchloric acid for polyamine determinations or in 50 mM sod. phosphate buffer, pH 7.2, containing 0.1 mM EDTA and 5 mM dithiothreitol for ODC assay.
Analytical Methods. Polyamines in perchloric acid extracts were measured by spectrophotofluorimetry of the dansylated adducts after se- paration by thin layer chromatography (11). ODC activity was assayed according to Djurhuus (12) as described previously (4). Protein was determined by the method of Lowry et al. (13).
45Ca Influx. Rates of 45Ca2+ influx were measured in synaptosomal fractions by a modification of the method of Drapeau & Blaustein (14) using PSS as incubation medium. Synaptosomal suspensions containing l-2 mg protein per ml were preincubated for 30 min at 37' C under 95% C2-5% CO2 in a Dubnoff shaker with additions as described in RESULTS. 100 pl aliquots containing 100-200 n$ of synaptosomal protein were pipetted onto prewashed glass fiber discs (GF/C-Whatman) in an Amicon VEM-2 filtra- tion apparatus. mately lo6
Uptake of 45Ca2+ was initiated by adding 45Ca2+ (approxi- cpm/ml) in PSS or depolarizing medium without and with other
additions. Incubations were stopped by rapidly diluting incubation media with 20 vol. of ice-cold PSS containing 1 mM LaC13 ("stop solution"), and were filtered with suction and washed rapidly with three 2 ml-aliquots of stop solution. Filters with washed synaptosomes were placed in vials containing 10 ml of Aquasol and the radioactivity counted by liquid scintillation spectrometry. To correct for nonspecific uptake, the 45Ca2+ content at zero time (i.e., filtration immediately after adding 45Ca2+ in stop solution) was subtracted from the values obtained after incubations. This nonspecific uptake amounted to less than 10% of the 45Ca2+ taken up in the control medium after a 1 min incubation.
45Ca2+ Efflux. Synaptosomes were suspended in PSS, equilibrated for 15 min at 37' C under 95% 02-5% CO2 with constant shaking in the presence or absence of appropriate additions and then preincubated for an additional 10 min with 45Ca2+ (approximately 106 cpmfml). Synapto- somes were quickly washed once and resuspended in the same medium. 100 ~1 aliquots of synaptosomes (containing about 200 ug of protein) were pipetted into the wells of an Amicon VFM-2 filtration apparatus fitted with glass fiber filters (GF/C, Whatman). Samples were washed three times with 3 ml of cold medium of the same composition. 45ca2+ release was initiated by the addition of 2 ml of PSS or depolarizing solution with and without additions. 45Ca2+ efflux was stopped by the addition of 2 ml of "stop solution" and the effluent collected by filtration with suction. 45Ca2+ efflux was expressed as percent of total radioactivity (radioactivity in filtrate and on filter) contained in the filtrate.
Release of Neurotransmitters. The release of neurotransmitters from synaptosomes was measured after preloading the synaptosomes with radioactively labeled GABA or norepinephrine under similar conditions used for 45Ca2' efflux determinations. Efflux of the incorporated neurotransmitters was measured by using the filtration procedure des-
565
Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cribed for 4sCa2+ effl ux except that cold PSS was used as the stop solu- tion.
Results
ODC Activity and Polyamine Concentrations. The ODC activity of
freshly isolated synaptosomes was similar to that of cerebral cortex
(~10 pmol/hr/mg of protein). Depolarization of synaptosomes with
high K+ (50 mM) induced an abrupt, transient increase in ODC activity
(Fig. 1A). ODC activity was increased nearly 2-fold at 15 set, reached
a maximum (3-fold) at 30 and 60 set, declined to near control values
at 3 min, and again increased after 30 min of incubation in the con-
A. PUTRESCINE
B 300
Time (minutes) limo Wnutes
Fig. 1. Potassium depolarization evokes an acute transient increase in ODC activity (A) and an early increase in polyamine concentrations (8) in rat cerebral cortex synaptosomes. Synaptosomes were incubated at 370 C with 50 z&l I@ for various times and analyzed for ODC activity and polyaminc levels as described in METHODS. The zero time control values for ODC was ll+ 1.6 pmoles/mg protein/h; and for polysmines (nmoles/mg protein) were: spermine, 0.85 + 0.053; spermidine 0.72 + 0.058 and putrescine, 0.35 + 0.038. The values are means + SEM (~4). *,**,***: pcO.05, 0.01, 0.001 (vs control).
566
Vol. 133, No. 2, 1985 BIOCHEMICAL AND BiOPHYSICAL RESEARCH COMMUNICATIONS
tinuous presence of 50 mM K+. All three polyamines also increased
rapidly following potassium depolarization, with the initial rate of
increase in putrescine exceedfng that of spermidine and spermine at
15 set (Fig. 1B). The putrescine and spermidine levels peaked at
1 min, and the spermine level at 3 min. Polyamine levels tended to
decline between 3 min and 30 min, but putrescine and spermine were
still significantly increased at 30 min. Fresh synaptosomes contained
Z- to 3-fold greater concentrations of putrescine and spermidine than
cerebral cortex, but spermine concentrations were similar. Synaptosomal
polyamine levels declined by 30 to 50% during 30 min preincubation at
37O c in PSS. DFHO, a specific enzyme-activated, irreversible inhibitor
of ODC (15), abolished the depolarization-induced increase in ODC
activity (not shown) and polyamine accumulation (Fig. 2). Indeed
synaptosomal polyamines were decreased to below basal values by DFMO in
the presence of high K' during a 60 see incubation. Putrescine (1 mM),
when added together with DFMO and high K+, circumvented the DFMO effect
and significantly increased the concentration of putrescine, spermidine
and spennine in synaptosomes (Fig. 3). These experiments show that an
A. PUTRESCINE 8. SPERMIDINE c. SPE~~MINE
+ 2. Fig. DE’MO blocks the potassium-stimulated increase of polyamides,
and putrescine nullifies the DFMO effect. Synaptosomes were preincubat- ed for 15 rain at 37O C with 5 mM DFMO, DFMO and 1 mM putrescine, or no addition and then were incubated with or without 50 mM IC+ for 1 min. Corrections for nonspecific uptake of putrescine were made by subtracting the O" C uptakes. Data are means + SEM (n=4). *,**,***: ~~0.05, 0.01, 0.001 (vs control). tt,ttf: p<O.El, 0.001 (vs 50 mU K+). 44 $9+: ~~0.01, 0.001 (vs DFMO + 50 mM I+).
567
Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
25
r A
IS 30 45
TIME (socondr)
20
15
IO
5
0
0 15 30
Time (seconds)
60
3. Fig. DFMO suppresses the potassium-induced stimulation of 45
Ca influx (A) and efflux B) in synaptosomes and putrescine antagonizes DEMO sup- pression. A. t 5Ca influx. Synaptosomes were preincubated with 5 mM DEMO and 1 mM putrescine or no a ent for 15 min at 37' C. At zero time, 5 mM K+ (0-e) and 50 mM 8 (0 ------- 0) was added to synapto- some8 preincubated with DEW0 (A -.-.-.-A), and DFMO and putrescine (A . . . . . A)), and 45Ca ($2 uCi/ml) was added to all tubes. Data are means + SEM(n=4). B. 45Ca efflux. Synaptosomes were preincubated with 5 mM DFMO, DFMO and 1 mM putrescine, or no addition for 15 min at 37' C. 45Ca (*Z uCi/ml) was then added, and the preincubation was terminated 10 min later. S without 45Ca. 4Y
aptosomes were washed and resuspended in the same medium Ca efflux was initiated by adding 5 mM K+ (0-O)
and 50 I&I K+ (O-----.0) to synaptosomes preincubated without an agent, and 50 mM K' to synaptosomes preintgbated with DFMO (r--.-.-r), and DFMO and putrescine (A...........4 . Ca efflux was terminated by the addi- tion of 2 ml of stop solution, and the effluxes and filter retentates were collected. Data are means + SEM (n=4). *x,*x*: pco.01, 0.001 (vs 5 mM K+). t,tt,ttt: pco.05, 0.81, 0.001 (vs 50 mM K+). +,++,+++: p<O.O5, 0.01, 0.001 (vs DFMO + 50 mM K+>.
568
Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
increase in ODC activity and augmented polyamine synthesis are very early
events in potassium depolarized synaptosomes.
Relation of Polyamine Synthesis to Ca2+ Fluxes. Since depolarizar
tion with high K+ is known to induce a rapid stimulation of Ca 2+ in -
flux in synaptosomes (1,2), we examined the role of polyamine synthesis
in this influx. Fig. 3 shows that high K+ evoked a substantial (2- to
3-fold) stimulation of 45Ca2+
influx into synaptosomes at 15 set (Fig.
3A), DFMO attenuated or abolished the depolarization-induced stimula-
tion of 45Ca2+ influx, and putrescine partially negated the DFWO effect.
Potassium depolarization also induced a large (3-fold) increase in
45Ca2+ efflux from 45Ca2+ -preloaded synaptosomes at 15 set (Fig. 3B).
DFMO suppressed the depolarization-induced efflux of 45Ca2+ , and putres-
tine nullified the effect of DFMO. These findings support the inference
that enhanced polyamine synthesis is involved in the stimulation of
Ca2+ influx and efflux in potassium depolarized synaptosomes.
Relation of Polyamine Synthesis to Neurotransmitter Release.
The role of polyamine synthesis in neurotransmitter release was
assessed in synaptosomes preloaded with L3H]GABA or t3R]norepinephrine.
Depolarization with high K' evoked a Ca 2+
-dependent, 2- to 3-fold
increase in the release of these neurotransmitters from synaptosomes
at 15 sec. DFMO suppressed the depolarization-induced stimulation of
[3H]GABA (Fig, 4) and [3R]norepinephrine (not shown) release, and
putrescine attenuated or nullified the DE'MO effect. These data pro-
vide strong evidence for the view that rapid polyamine synthesis is
involved in the Ca 2f
-dependent release of neurotransmitters from
synaptosomes depolarized by high k.
Discussion
Synaptosomes retain most of the functional properties of the
presynaptic nerve terminals from which they are derived, and thus pro-
vide a valuable system for the study of the molecular mechanisms
which couple Ca 2+ fluxes to neurotransmitter release (1,2). The pre-
569
Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0 15 30 50
Tlmc (seconds)
4. Fig. DFMO abolishes the potassium-induced stimulation of [3H]G~A release from synaptosomes and putrescine negates the DFMO effect, The experimental conditions were as described in Fig. 3B except that the synaptasomes were Incubated with [3~1~~~~ instead of [45Ca2+]. Data are means + SEM (1x=4).
sent study confirms previous reports demonstrating the presence of
putrescine, spermidine, spermine (16,171 and ODC (18) in rat brain
synaptosomes. However, the major finding to emerge from these ex-
periments is that potassium-induced depolarization of the synaptosomal
plasma membrane induces an early (~15 set), transient stimulation of
ODC activity and a concomitant rise in the concentration of putrescine,
spermidine and spermine in synaptosomes. ODC converts ornithine to
putrescine and is the first and rate-regulating enzyme in the polyamine
synthetic pathway (19). The kinetics of the potassium-stimulated ODC
and polyamine changes are consistent with the inference that the rapid
accumulation of polyamines resulted from ehnanced synthesis, rather
than reduced catabolism. Thus the rank order of the initial increase
of these constituents, expressed as percentage change at the earliest
measured time point (15 set) after addition of high K+, was ODC >
putrescine > spermidine = spermine. Moreover, putrescine and spermi-
dine peaked earlier (<l min) than spermine (<3 min). Further evidence
of ODC-initiated polyamine synthesis comes from the finding that DEMO
blocked the potassium-induced accumulation of polyamines. These ob-
servations imply that all of the enzymes and substrates of the polyamine
570
Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
synthetic pathway are present in synaptosomes. However, only S-adenosyl-
l-methionine decarboxylase (18) and ODC (18; and this paper) have been
demonstrated in synaptosomes to date.
The rapid formation of polyamines in potassium-depolarized synapto-
somes suggests that polyamines may serve as second messengers for
stimulus-secretion coupling in presynaptic nerve terminals. We there-
fore evaluated the polyamine dependence of potassium-stimulated 4SCa2+
fluxes and t3H]GABA and [3H]norepinephrine release with the aid of DFMO,
a suicide inhibitor of ODC which has not other known biological effect
(15,lY). DFMO suppressed the potassium-evoked accumulation of synapto-
somal polyamines and concurrently attenuated or blocked the stimulated
increment in 45Ca2+ influx and efflux and neurotransmitter release.
Added putrescine augmented DPMO-depleted polyamine stores and attenuated
or abrogated DFMO suppression of 4SCa2f fluxes and neurotransmitter re-
lease in depolarized synaptosomes. Apparently putrescine is transported
into synaptosomes and partly converted to spermidine and spermine. These
findings strongly suggest that rapid polyamine synthesis is essential
for the mediation of enhanced Ca 2+
fluxes and neurotransmitter release
in potassium-depolarized synaptosomes.
Potassium-stimulated Ca 2+ entry into presynaptic nerve terminals
appears to occur through two separate processes with distinct time courses
and ionic and voltage sensitivities, referred to as fast-phase and slow-
phase uptake (14,ZO). The fast pathway lasting l-2 set is thought to
modulate the phasic release of neurotransmitter evoked by an action
potential, whereas the slow pathway may modulate transmitter release after
longer lasting depolarizations, e.g., tetanic stimulation (20). While it
appears unlikely that the stimulation of ODC activity and polyamine syn-
thesis in potassium-depolarized nerve endings can occur rapidly enough
to account for the fast phase of Ca 2f influx, the time course of this
stimulation correlates well with the slow-phase of Ca 2+ influx and Ca2'-
dependent transmitter release. Potassium depolarization also evokes an
571
Vol. 133, No. 2. 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
early (cl5 set) increase in Ca 2+ efflux indicating mobilization of Ca 2+
influx and Ca2+ -dependent transmitter release. Potassium depolarization
also evokes an early (cl5 set) increase in Ca 2+ efflux indicating
mobilization of Ca 2+
from intracellular storage sites. Therefore, the
increase in cytosolic Ca 2+ concentration occurring in potassium de-
polarized synaptosomes (21-23) is a consequence of enhanced Ca 2+ influx
and redistribution of internal Ca 2+
.
The transient depolarization- or voltage-dependent changes in ODC
activity in synaptosomes presumably represents an activation-deactivation
cycle of a cryptic ODC via a posttranslational process, possibly in-
volving a oovalent modulation of ODC or an ODC regulatory protein
such as ODC antizyme (19). These changes in ODC activity and polyamine
synthesis closely resemble those observed in target cells following re-
ceptor activation by hormones and other agonists (4-7,24). In receptor-
mediated responses, as in potassium-depolarized synaptosomes, stimula-
tion of Ca 2+ fluxes and Ca2+ -mediated cell processes has been found to
be sensitive to DFMO and this DFMO sensitivity can be negated by putres-
tine. These findings are consistent with a model for signal transduction
and stimulus-response coupling in which a rapid, short-lived stimulation
of the catalytic activity of a latent ODC in or near the plasma membrane
is a key reaction. Newly synthesized polyamines serve as messengers or
signals which mediate cellular responses by enhancing Ca 2+ influx and/or
mobilizing internal Ca 2+ , leading to a rise in cytosolic Ca 2+ concentra-
tion. Polyamine signals may activate Ca 2+ channels by displacing Ca 2+
bound to channel molecules, resulting in a conformational change in the
Ca2+ channel, and may mobilize internal Ca 2+ by releasing Ca 2+ bound to
acidic sites in the plasma membrane, mitochondria and endoplasmic reti-
culum via a cation-exchange reaction,
These findings are the first indication that ODC and polyamines
may have an important role in the transduction and transmission of
572
Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
voltage-mediated signals and in stimulus-secretion coupling in nerve
cells.
Acknowledgements
We thank Dr. J.J. Trout for the electron microscopy and Mrs. T.
Howell for secretarial work, This work was supported by the Veterans
Administration and NIH grants NS 18047 and HL 26835.
1.
2. 3. 4. 5.
6.
7.
8.
9. 10. 11.
12. 13.
14. 15.
16. 17. 18. 19. 20. 21.
22.
23.
24.
References
Kelly, R.B., Deutsch, J.W., Carlson, S.S. 6 Wagner, J.A. (1979) Ann. Rev. Neurosci. 2, 399-446. Reichardt, L.F. & Kelly, R.B. (1983) Ann. Rev. Biochem, 52, 871-926. Henkart, M. (1980) Fed. Proc. 3V, 2783-2789. Koenig, H., Koenig, H.,
Goldstone, A.D. & Lu, C.Y. (1983) Nature 305, 530-534. Goldstone, A.D. 6 Lu, C.Y. (1983) Proc. Nat. Acad. Sci.
